Supporting Information

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Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11
Supporting Figure 12
Supporting Table 1
Supporting Table 2

Fig. 8. Sohlh1 structure. (A) Sohlh1 nucleic acid (GenBank accession no. AY623913) and SOHLH1 amino acid sequence is shown. Nucleotides and amino acids corresponding to the basic helix-loop-helix domain are underlined. The presumed initiator methionine and stop codon are boxed. (B) Comparison of the basic helix-loop-helix domain among human, chimpanzee, mouse, and rat SOHLH1.

Fig. 9. Sohlh1 RNA expression. (A) Multitissue RT-PCR with oligonucleotides specific for Sohlh1 and actin. cDNA was generated from total RNA extracted from adult heart, liver, lung, brain, spleen, kidney, uterus, testis, intestine, and ovary. (B) Sohlh1 expression in the WT embryonic ovaries collected at E13.5, 14.5, 15.5, 17.5, and 18.5. Actin expression in embryonic gonads is also shown.

Fig. 10. Expression of Figla and the zona pellucida genes 1-3 in newborn WT and Sohlh1^-/- ovaries. Quantitative RT-PCR is shown for relative expression of Figla (A), Zp1 (B), Zp2 (C), and Zp3 (D). Figla, Zp1, and Zp3 show statistically significant down-regulation in mutant ovaries. Only Zp2 does not show a statistically significant decrease in expression in the mutant ovaries as compared with WT. Data are normalized to Gapd expression and presented as the mean relative quantity (compared to WT), with error bars representing the SEM. Student’s t test was used to calculate P values. *, P < 0.05; **, P < 0.01.

Fig. 11. Expression of oocyte-expressed genes in newborn WT and Sohlh1^-/- ovaries. (A-F) Relative expression of Nobox (A), Gdf9 (B), Pou5f1 (C), Nohma (D), Stra8 (E), and Lhx8 (F) examined by quantitative RT-PCR. Expression in the knockout is relative to expression in the WT. (G) Analysis by RT-PCR shows that the genes down-regulated in Nobox^-/- are also down-regulated in the Sohlh1^-/- (-/-) ovary. *, P < 0.05; **, P < 0.01.

Fig. 12. E box elements in promoters in Lhx8, Nobox, Zp1, Zp2, and Zp3 for luciferase reporter constructs and ChIP experiments. (A) Multiple species alignments are shown. Conserved E box sequences are shown underlined, and positions relative to the transcriptional start site are indicated. (B) PCR amplification of promoter regions is shown for the ChIP assay. "P1" is the PCR product encompassing the indicated E box in the promoter. "C1" is a control PCR product within the coding region of the target genes.

Table 1. Oligonucleotides used to generate plasmids and mutants

Added enzyme sites are boldface, and mutated sequences are underlined.

Table 2. Oligonucleotides used for chromatin immunoprecipitation